The endocannabinoid system is the signaling network in the brain hijacked by the active component of marijuana, delta-9 tetrahydrocannibinol (THC), which elicits its effects primarily through activation of the central cannabinoid receptor (CB1) in the brain. The endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) are the endogenous ligands of CB1 and are thought to modulate a diverse array of physiological processes including appetite, pain, emotions, cognition and addiction. Endocannabinoid levels in vivo are tightly regulated by enzymatic biosynthesis and degradation and, as such, these enzymes are principle regulators of EC signaling and tone. Degradation of AEA by the integral membrane protein fatty acid amide hydrolase (FAAH) has been well-characterized in vivo through the use of FAAH knockout mice and FAAH selective inhibitors. In contrast, the enzymes responsible for 2-AG termination have not been fully characterized. Using a functional proteomics approach, we identified three enzymes which collectively perform >98% of the 2-AG hydrolase activity in mouse brain proteomes: monoacylglyceride lipase (MAGL), and the uncharacterized alpha/beta hydrolases 6 and 12 (ABHD6 and ABHD12). We propose to biochemically characterize the substrate selectivity, kinetic parameters and cellular/subcellular localization of recombinant preparations of MAGL, ABHD6 and ABHD12 and generate mouse models bearing targeted disruptions of these enzymes using genetic and pharmacological means. The results of these studies should allow the elucidation of the relative roles of these enzymes in endocannabinoid signaling and might also identify promising drug targets for therapeutics that elicit some of the medicinal benefits of THC without the psychoactive side-effects. The endocannabinoid 2-arachidonoylglycerol (2-AG) is a lipid neurotransmitter thought to mediate many (patho)physiological processes including obesity, pain, anxiety, depression and addiction. The goal of the proposed research is to elucidate the mechanisms by which 2-AG mediated signaling is terminated. The results of this study will increase the basic understanding of the endocannabinoid system and may also identify promising drug targets for the treatment of human diseases regulated by the endocannabinoid system.